The project objective is to clone and express selected toxin components from Bordetella pertussis. Of particular interest in pertussis toxin (PT) or "pertussigen" a multifunctional pathogen consisting of a protein complex of 5-6 nonidentical subunits. The biological function of these subunits has not been established. The primary methods employed are oligonucleotide screening of molecular cloned pertussis DNA and direct gene cloning-expression vector system. These two parallel approached will result in the identification and isolation of the portion of DNA coding for the pertussis toxin. Using these data and DNA clones, genetic manipulation of the pertussis genome may result in a highly immunogenic, nontoxic antigen which could lead to the development of a safer pertussis vaccine. Oligonucleotide Screening. Pertussis toxin was fractionated into subunits using high pressure liquid chromatography. Two of the subunit polypeptides were partially sequenced and, based on this amino acid sequence, two oligonucleotide DNA probes were synthesized. These DNA probes are currently being used to screen fragments of the pertussis genome DNA. Once these fragments are identified, adjacent fragments can be located and sequenced. Computer analysis of the DNA sequence will reveal the molecular structure of the "transcriptional unit" and identification of regulator sites may be possible. These identified fragments will then be used to genetically manipulate the pertussis genome. Direct Cloning-expression System. A cloned library of pertussis DNA fragments will be tested in a direct expression vector system. Antibodies against pertussis toxin will be employed to identify those DNA fragments which produce polypeptides recognized by the antibody. Polyclonal and monoclonal antibodies have been developed in our laboratory which specifically recognize pertussis toxin components in a natural and denatured state; these will be useful in the expression screening. Currently, we are screening the monoclonal antibodies to see what effect they have on the biofunction of the toxin.